step i: white paper application - craig venter · 2020-01-27 · name vishnu chaturvedi position...

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Genomic Sequencing Centers for Infectious Diseases: White Paper Form 1

Step I: White Paper Application

Application Guidelines

1. The application should be submitted electronically per NIAID’s requirements (TBD).

Include all attachments, if any, to the application.

2. GSC personnel at any of the three Centers can assist / guide you in preparing the

white paper.

3. Investigators can expect to receive a response within 4-6 weeks of the review cycle.

4. Financial agreements would require appropriate justification and approval; payment

arrangements need to be established in advance with the GSC.

5. Upon approval of the white paper concept, the NIAID Project Officer will assign the

project to an NIAID GSC to develop a Management Plan in conjunction with the

participating scientists.

Please Note: This is a draft template. Users may have to adapt contents to a final template NIAID develops in the coming weeks and adhere to a process that will be established for submission and approval of projects; it shall be communicated once finalized.

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White Paper Application

Project Title: Cryptococcus gattii Genomics and Transcriptomics

White Paper Submission Date (08/07/10):

Principal Investigator Contact:

Name Vishnu Chaturvedi

Position Director, Mycology Laboratory

Institution Wadsworth Center, New York State Department of Health

Address 120 New Scotland Ave.

Albany, NY 12208

Telephone 518-474 4177

Fax 518-486 7971

E-Mail [email protected]

Co-Principal Investigator Contact:

Name K.J. Kwon-Chung

Position Chief, Molecular Microbiology Section

Institution Laboratory of Clinical Infectious Diseases, National Institute of

Allergy and Infectious Diseases

Address Bldg. 10, Room 11N234, NIH

Bethesda, MD 20892

Telephone 301-496-1602

Fax 301-480-3240



Name John Perfect

Position Interim Chief, Division of Infectious Diseases

Institution Department of Medicine, School of Medicine, Duke University

Address DUMC 3353

Durham, NC 27710


Fax 919-684-8902



Name Brian Wong

Position Director, Division of Infectious Diseases

Institution Oregon Health & Science University

Address 3181 SW Sam Jackson Park Road L457

Portland OR 97239-3098


Fax 503-494-4264


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All white papers will be evaluated based on the following sections.

1. Executive Summary (Please limit to 500 words.)

Provide an executive summary of the proposal.

Cryptococcus gattii is an emerging global pathogen that was first recognized in 1970 from

Central Africa. Earlier, C. gattii CNS, lung and skin infections were believed to be limited

to healthy individuals in tropics and subtropics. Early investigations suggested that these

infections were not seen in HIV-AIDS patients. These perspectives might have led to less

attention being paid to C. gattii vis-à-vis C. neoformans, which has received maximum

attention as an opportunistic pathogen in HIV-AIDS patients. However, many recent

reports suggest that C. gattii cryptococcosis is more common in immunocompetent as well

as HIV-AIDS patients globally; this is matched by isolations of fungus from more than 50

tree species in temperate and tropical areas. More recently, C. gattii infections and deaths

in the Pacific Northwest and Vancouver, Canada have raised public health awareness in

North America.

Cryptococcus gattii cryptococcosis has more pulmonary and cerebral nodules

(cryptococcomas), increased neurological morbidity, and slower response to treatment vis-

à-vis C. neoformans disease. Limited studies have indicated that outstanding questions

regarding C. gattii cryptococcosis cannot be resolved by extrapolation from C. neoformans

pathogenic factors. We do not know the reasons and significance of the disparity in the

global distribution of C. gattii serotype B and C strains. The majority of isolates from

Canada are serotype B, MAT, VGIIa genotype and the emergence of a ‘hypervirulent

clone’ was implied to be the reason for the outbreak. However, this is not entirely tenable

since a similar strain C. gattii NIH444 (serotype B, VGIIa, MAT) was isolated from

sputum of a patient in 1970 from Seattle, WA. Beside, genotypes VGI, VGIII and VGIV

and serotype C have been reported from patients from other areas. Thus, we have few

clues as to what causes emergence or reemergence of highly pathogenic strains, why C.

gattii split up from its pigeon guano dwelling sibling C. neoformans, and why it chose to

climb into trees instead, and why immunocompetent individuals are vulnerable to this

pathogen and what can be done to prevent the infection?

The information on the genome of C. gattii is inadequate and unrepresentative. Currently,

draft genome sequences are available for two strains, R265 and WM276, which are MAT,

serotype B, genotype VGII/VGI from Canada and Australia, respectively. There is a gap

in knowledge about the genomes of VGIII and VGIV strains, serotype C strains, and

MATa strains. The geographical representation is inadequate in the absence of strains from

California, South America, Asia and Africa. Additional obstacles are: a) complex

molecular typing schemes, and b) lack of functional analyses (transcriptomics) that can

foretell C. gattii response under pathogenic and non-pathogenic conditions.

We propose complete genome sequencing of 12 reference strains by next generation

sequencing technology. Transcriptomes of 12 C. gattii strains will also be mapped by

RNA-Seq technology. The selection of strains, sampling conditions, analytical strategies

and data release plan were designed with input from the 3-Co-PIs, and additional

researchers representing global C. gattii community. The project would provide: 1) insight

into C. gattii genomes to anchor future research studies, 2) validation of single nucleotide

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polymorphisms (SNPs) for molecular typing to improve epidemiology studies, 3) transcript

analyses to fast forward the discovery of proteins for diagnostics, drug targets and

vaccines. Overall, the project will fill-in important gaps in our knowledge about an

important emerging pathogen, accelerate research efforts and translate laboratory findings

into tangible public health benefits.

2. Justification

Provide a succinct justification for the sequencing or genotyping study by describing

the significance of the problem and providing other relevant background information.

Public health significance.

C. gattii cryptococcosis is an emerging global problem: C. gattii is an emerging pathogen

that has triggered serious public health concerns due to (i) its appearance in previously

unknown geographic areas, (ii) its outbreaks among healthy humans, pets, and wildlife,

(iii) the intractable nature of cryptococcal disease, and (iv) problem with routine diagnosis

in clinical laboratories. (1, 2, 9, 16, 25, 28, 44, 45, 51, 54, 56). Initial studies suggested

that HIV-AIDS patients rarely get infected with C. gattii, but more recent publications

indicate a higher prevalence of C. gattii infections in both immunocompetent hosts and

HIV-AIDS patients (9, 12, 23, 29, 40, 42, 67).

Global burden of C. gattii disease remains underestimated: There is an obvious disconnect

between natural isolations of C. gattii and its finding in clinical materials in some areas

while the converse is true in other areas (65). Reliable estimates for rates of

cryptococcosis due to C. gattii are currently lacking, but one estimate suggests that one-

third to one-tenth of cryptococcosis cases worldwide are caused by C. gattii(20, 27, 49, 55,

63).

C. gattii natural habitat(s) poses occupational and recreational risks: C. gattii natural

habitat is believed to be on a variety of trees especially in tree-hollows (19, 38, 65). C.

gattii poses travel-related risks to humans and animals traveling to the areas considered to

be ‘hot spots’ for the natural occurrence of the fungus.(6, 7, 25, 39, 47). Environmental

exposure due to occupational or recreational activities increases the risks for acquisition of

infections especially among males who are over-represented among affected populations

(18, 30, 31).

C. gattii cryptococcosis has many complications and is difficult to treat: C. gattii

cryptococcosis has more complicated course than C. neoformans cryptococcosis. The

complications include more pulmonary and cerebral nodules (cryptococcomas), persistent

mental status abnormalities, increased neurological morbidity, surgical interventions and

slower response to treatment vis-à-vis C. neoformans disease (15, 17, 24, 57-59, 62, 64).

C. gattii strains have higher level of heteroresistance to fluconazole: C. gattii strains

exhibit relatively higher levels of heteroresistance to fluconazole (LHF) as compared to C.

neoformans strains (61, 70). This phenomenon refers to variable intrinsic resistance

exhibited by C. gattii strains to fluconazle, which is the most common drug used for the

treatment of cryptococcosis. LHF could be an important determinant of outcome in C.

gattii cryptococcosis. The mechanism of action of LHF remains undetermined since prior

exposure to the drug is not a pre-requisite for the drug resistance.

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Many diagnostic laboratories are unprepared to report C. gattii infections: The serotype

variations in C. neoformans species complex and the existence of serotypes B and C,

which would define future C. gattii, were known as early as 1950 (21). However, the wide-

spread use of serotyping system became possible in diagnostic laboratories only after the

reagents became commercially available (26). Alas, this reagent is no longer available and

no serotyping is being carried out anymore. Similarly, selective isolation medium

(nigerseed agar) and differentiation medium for C. neoformans and C. gattii are well

described, but these require specialized reagents and therefore, they are also not readily

available in most diagnostic laboratories (37, 66). Thus, there is a pressing need to find a

facile method for routine identification of C. gattii in diagnostic laboratories.

Great heterogeneity in C. gattii isolates points to possible sub-species: The currently

recognized serotypes (B, C) and genotypes (VGI-VGIV) do not fully represent the great

heterogeneity in C. gattii strains isolated from clinical and environmental sources (52). The

recent emergence of this infection in Pacific Northwest and Vancouver, Canada has

revealed unique pathogenic attributes of sub-genotypes within VGII strains (5, 32). The

information is also starting to emerge about similar association between other sub-

genotypes and distinct virulence properties.

Host immune responses critical for C. gattii infection and recovery are not understood: A

few surveys in high incidence areas have revealed many seropositive individuals, which

suggest that the burden of exposure is much larger than can be estimated from the people

with apparent disease (60). Thus, not all exposed individuals develop C. gattii infections

and there are host factors like HLA types that are critical for the control and elimination of

this pathogen (68). Conversely, there are yet undefined predisposition events that cause

apparently healthy individuals to suffer from C. gattii infections (51). Preliminary data

indicates a role for protective inflammation in controlling this disease (4, 11).

Apparently unique pathogenic attributes of C. gattii remain poorly defined: Both C. gattii

and C. neoformans share obvious virulence characteristics such as capsule, thermal

tolerance and laccase. However, C. gattii is suspected to prevent immune cells in the lungs

from mounting effective defense, but we do not know about mechanisms behind such an

exquisite strategy. Similarly, a number of proteins relevant to the virulence in C.

neoformans are employed by C. gattii in apparently distinct manner and more in depth

investigations in this area are needed.

Genomics tools are needed to find better targets for diagnostics, drugs and vaccine: A

custom-designed C. gattii whole-genome tiling microarray was recently used to discover

an important role for mitochondrial parasitism in fungal virulence (46). However, the poor

annotation of existing C. gattii genomes prevented these investigators from full

identification of other significant virulence genes in C. gattii R265. Due to better genome

annotations, the transcriptomic analyses are far more advanced in C. neoformans and have

revealed important insights into oxygen and CO2 sensing mechanisms, capsule synthesis,

and cyclic AMP signaling (8, 13, 14, 33, 48). Clearly, whole genome profiling tools are

needed for C. gattii for advancing our understanding of this enigmatic pathogen and for

translating such knowledge for public health benefit.

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3a. Rationale for Strain Selection

The strains proposed to be included in genome sequencing and/or transcriptomics are

summarized in Table 1; accompanying details provide rationale for their selection.

Table 1. List of C. gattii strains to be sequenced.

Rationale for selection

C. gattii RV20186

This is the type strain of C. gattii originally isolated from a seven-year old boy from the

Democratic Republic of Congo who was successfully treated for cryptococcal meningitis.

The CSF culture yielded two colony types – R and B, which were also reproduced when

fungal cells were injected in mice. Although the case was diagnosed in 1966, this region is

believed to have many similar infections in young men dating back to early 1950s and 60s.

Thus, this strain could provide an invaluable window to the earliest known infections and a

reference for tracking evolutional history and population biology of C. gattii.

No. Strain Source/Origin Characteristics Selection Criteria Ref.

1 RV20186 CSF / Democratic Republic of Congo

VGI/ Serotype B Type strain of C. gattii (3, 69)

2 WM179 Human/Australia VGI/ Serotype B/

MAT

Pathogenic in a rat model of C. gattii cryptococcosis

(35, 50)

3 LA295 Human/ Argentina VGIIa/ Serotype B/

MAT

More pathogenic than R265- the major genotype in Vancouver outbreak

(53)

4 R272 Human/ Canada VGIIb / Serotype B/

MAT

‘Minor’genotype in Vancouver outbreak; low pathogenic potential

(22)

5 EJB18 Human /Oregon VGIIc /Serotype B/

MAT

Novel genotype from Oregon outbreak; highly pathogenic in mice

(5)

6 WM198 (McBride)

Feline/Australia VGIIb/Serotype B/

MAT

Veterinary strain, reactivation disease, low virulence in mice, high virulence in rat

(34, 35)

7 NIH444 CSF /Washington VGIIa /Serotype B/

MAT MAT Type strain of F. bacillispora; Host strain for pathogenesis studies

(10, 36)

8 VMGc3 Cactus plant/ Puerto Rico

VGII / Serotype B/ MATa

Environmental MATa strain (43)

9 NIH191 CSF/ California VGIII (AFLP 5C)/ Serotype C/ MATa

MAT a Type strain of Filobasidiella bacillispora (Sexual form)

(36)

10 USC1499 CSF/ California Genotype VGIII

/Serotype B/ MAT

Highly pathogenic strain from AIDS patient

(9)

11 Bt12 CSF/Botswana VGIV/ Serotype C/

MAT

Genetic identity to Serotype C strains from Botswana and Malawi

(41)

12 IND107-97

Eucalyptus/India VGIV / Serotype B Environmental strain /low fluconazole heteroresistance

(70)

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C. gattii WM179

This is a human strain from Australia, which represents relatively rare VGI genotype. It

has been found to be pathogenic in a rat model of C. gattii. This will be a crucial for

comparative pathogenic studies.

C. gattii LA295

This human strain originated in Argentina. It belongs to VGIIa, the major hypervirulent

genotype implicated in recent outbreak of C. gattii disease in Vancouver, British

Columbia, Canada. Importantly, LA295 is more pathogenic than R265 – the type strain

analyzed from the Canadian outbreak.

C. gattii R272

This human strain originated from Canada. It is genotype VGIIb, the minor genotype in

the Canadian outbreak and reportedly with low pathogenic potential.

C. gattii EJB18

This human strain represents a novel genotype – VGIIc, which is predominant is Oregon

outbreak. It is also highly pathogenic in mice models.

C. gattii WM198 (McBride)

This veterinary strain is VGIIb genotype. It has low virulence in mice, but high virulence

in rat model. Moreover, it is the only strain that has proven to be able to be reactivated in

animal after full treatment more than 10 years.

C. gattii NIH444

This VGIIa strain isolated from sputum of a patient from Seattle in 1970 is very similar to

R265, the outbreak strain from Canada. It has been shown to be an optimal host strain for

many experimental pathogenesis studies.

C. gattii VMGc3

This is a rare VGII, MATa environmental strain from Puerto Rico. This is one of the two

MATa strains included in this study to compare with remaining MAT strains.

C. gattii NIH191

This is the type strain of the sexual state of C. gattii termed Filobasidiella bacillispora.

This is a clinical strain with three unusual characteristics- serotype C, MATa and VGIII

genotype. It presents unique opportunities for genomic and phenotypic comparisons.

C. gattii USC1499

This is a clinical strain from HIV-AIDS patient from California. It has a VGIII genotype

and high virulence for mice similar to a hypervirulent VGIIa strain involved in recent

outbreak from Vancouver Island, British Columbia. It has been characterized extensively

for its phenotypes and genotypes. It could provide better understanding of the

pathogenesis of yet to be investigated VGIII strains. The choice is also relevant from the

perspective of the earliest known geographical distribution of C. gattii in Southern

California.

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C. gattii Bt12

This strain has been isolated from a patient in Southern Africa. The two rare characteristics

are serotype C and VGIV genotype. It has been used in genetic analyses that revealed a

lack of genetic diversity in clinical strains from Botswana and Malawi; these two countries

have high incidence of HIV-AIDS.

C. gattii IND107-97

This is an environmental strain, rare VGIV genotype and geographically diverse (India).

Another interesting feature is low level of fluconazole heteroresistance.

Environmental, Human and Veterinary Strains

VMGc3, IND107-97, WM179 LA295, R272, EJB18, WM198, NIH444 provide widest

possible diversity in clinical and environmental sources of C. gattii strains. These strains

have been the subject of preliminary studies and this set would be optimal in

transcriptomics to unravel changes in global expression in strains encountered in nature

and in disease.

Strains for pathogenesis studies

NIH444, USC1499, WM179, WM198 are strains that would be very useful for molecular

pathogenesis studies as they have stable differences in virulence, measurable putative

virulence factors that could be manipulated by gene knockouts and good animal models.

Transcriptomics on these strains would facilitate a correlation of virulence with genome-

wide expression of known and yet to be recognized virulence traits.

Strains for fluconazole heteroresistance analysis

C. gattii exhibits high level of fluconazole heteroresistance (LHF) with direct correlation

with high virulence. By implication, fluconazole heteroresistance could be an important

factor behind the poor therapeutic responses seen in patients treated with standard regimen

of antifungal drugs. Two high–low heteroresistance strains (NIH444 and IND107-97)

would provide an ideal set for transcriptomic analysis to understand the mechanisms

behind the observed resistance.

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Genomic Sequencing Centers for Infectious Diseases: White Paper Form

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Brazil. Rev Inst Med Trop Sao Paulo 50:75-78.

41. Litvintseva, A. P., R. Thakur, L. B. Reller, and T. G. Mitchell. 2005. Prevalence

of clinical isolates of Cryptococcus gattii serotype C among patients with AIDS in

Sub-Saharan Africa. J Infect Dis 192:888-92.

42. Lizarazo, J., M. Linares, C. de Bedout, A. Restrepo, C. I. Agudelo, and E.

Castaneda. 2007. [Results of nine years of the clinical and epidemiological survey

on cryptococcosis in Colombia, 1997-2005]. Biomedica 27:94-109.

43. Loperena-Alvarez, Y., P. Ren, X. Li, D. J. Bopp, A. Ruiz, V. Chaturvedi, and

C. Rios-Velazquez. Genotypic Characterization of Environmental Isolates of

Cryptococcus gattii from Puerto Rico. Mycopathologia.

44. Lopez-Martinez, R., J. L. Soto-Hernandez, L. Ostrosky-Zeichner, L. R.

Castanon-Olivares, V. Angeles-Morales, and J. Sotelo. 1996. Cryptococcus

neoformans var. gattii among patients with cryptococcal meningitis in Mexico.

First observations. Mycopathologia 134:61-4.

45. Lui, G., N. Lee, M. Ip, K. W. Choi, Y. K. Tso, E. Lam, S. Chau, R. Lai, and C.

S. Cockram. 2006. Cryptococcosis in apparently immunocompetent patients. Qjm

99:143-51.

46. Ma, H., F. Hagen, D. J. Stekel, S. A. Johnston, E. Sionov, R. Falk, I. Polacheck,

T. Boekhout, and R. C. May. 2009. The fatal fungal outbreak on Vancouver

Island is characterized by enhanced intracellular parasitism driven by mitochondrial

regulation. Proceedings of the National Academy of Sciences 106:12980-12985.

47. MacDougall, L., S. E. Kidd, E. Galanis, S. Mak, M. J. Leslie, P. R. Cieslak, J.

W. Kronstad, M. G. Morshed, and K. H. Bartlett. 2007. Spread of Cryptococcus

gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA.

Emerg Infect Dis 13:42-50.

48. Maeng, S., Y. J. Ko, G. B. Kim, K. W. Jung, A. Floyd, J. Heitman, and Y. S.

Bahn. 2010. Comparative transcriptome analysis reveals novel roles of the Ras and

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cyclic AMP signaling pathways in environmental stress response and antifungal

drug sensitivity in Cryptococcus neoformans. Eukaryot Cell 9:360-78.

49. McCarthy, K. M., J. Morgan, K. A. Wannemuehler, S. A. Mirza, S. M. Gould,

N. Mhlongo, P. Moeng, B. R. Maloba, H. H. Crewe-Brown, M. E. Brandt, R.

A. Hajjeh, and for the Gauteng Cryptococcal Surveillance Initiative Group.

2006. Population-based surveillance for cryptococcosis in an antiretroviral-naive

South African province with a high HIV seroprevalence. AIDS 20:2199-2206.

50. Meyer, W., D. M. Aanensen, T. Boekhout, M. Cogliati, M. R. Diaz, M. C.

Esposto, M. Fisher, F. Gilgado, F. Hagen, S. Kaocharoen, A. P. Litvintseva, T.

G. Mitchell, S. P. Simwami, L. Trilles, M. A. Viviani, and J. Kwon-Chung.

2009. Consensus multi-locus sequence typing scheme for Cryptococcus

neoformans and Cryptococcus gattii. Med Mycol 47:561-70.

51. Morgan, J., K. M. McCarthy, S. Gould, K. Fan, B. Arthington-Skaggs, N.

Iqbal, K. Stamey, R. A. Hajjeh, and M. E. Brandt. 2006. Cryptococcus gattii

infection: characteristics and epidemiology of cases identified in a South African

province with high HIV seroprevalence, 2002-2004. Clin Infect Dis 43:1077-80.

52. Ngamskulrungroj, P., F. Gilgado, J. Faganello, A. P. Litvintseva, A. L. Leal,

K. M. Tsui, T. G. Mitchell, M. H. Vainstein, and W. Meyer. 2009. Genetic

diversity of the Cryptococcus species complex suggests that Cryptococcus gattii

deserves to have varieties. PLoS One 4:e5862.

53. Ngamskulrungroj, P., C. Serena, F. Gilgado, R. Malik, and W. Meyer. 2010.

Global VGIIa isolates are of comparable virulence to the major fatal Cryptococcus

gattii Vancouver Island outbreak genotype. Clin Microbiol Infect.

54. Oliveira, F. M., C. B. Severo, L. S. Guazzelli, and L. C. Severo. 2007.

Cryptococcus gattii fungemia: report of a case with lung and brain lesions

mimicking radiological features of malignancy. Rev Inst Med Trop Sao Paulo

49:263-5.

55. Park, B. J. a., K. A. b. Wannemuehler, B. J. c. Marston, N. d. Govender, P. G.

e. Pappas, and T. M. a. Chiller. 2009. Estimation of the current global burden of

cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23:525-530.

56. Pérez, C., M. Dolande, M. Moya, A. Roselló, C. R. H. de Capriles, M. E.

Landaeta, and S. Mata-Essayag. 2008. Cryptococcus neoformans, Cryptococcus

gattii: Serotypes in Venezuela. Mycopathologia 166:149-153.

57. Perfect, John R., William E. Dismukes, F. Dromer, David L. Goldman,

John R. Graybill, Richard J. Hamill, Thomas S. Harrison, Robert A. Larsen,

O. Lortholary, M.-H. Nguyen, Peter G. Pappas, William G. Powderly, N.

Singh, Jack D. Sobel, and Tania C. Sorrell. 2010. Clinical practice guidelines for

the management of Cryptococcal disease: 2010 Update by the Infectious Diseases

Society of America. Clinical Infectious Diseases 50:291-322.

58. Phillips, P., K. Chapman, M. Sharp, P. Harrison, J. Vortel, T. Steiner, and W.

Bowie. 2009. Dexamethasone in Cryptococcus gattii central nervous system

infection. Clin Infect Dis 49:591-5.

59. Pukkila-Worley, R., and E. Mylonakis. 2008. Epidemiology and management of

cryptococcal meningitis: developments and challenges. Expert Opin Pharmacother

9:551-60.

60. Seaton, R. A., A. J. Hamilton, R. J. Hay, and D. A. Warrell. 1996. Exposure to

Cryptococcus neoformans var. gattii--a seroepidemiological study. Trans R Soc

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Trop Med Hyg 90:508-12.

61. Sionov, E., Y. C. Chang, H. M. Garraffo, and K. J. Kwon-Chung. 2009.

Heteroresistance to Fluconazole in Cryptococcus neoformans Is Intrinsic and

Associated with Virulence. Antimicrob. Agents Chemother. 53:2804-2815.

62. Soares, B. M., D. A. Santos, L. M. Kohler, G. da Costa Cesar, I. R. de

Carvalho, M. dos Anjos Martins, and P. S. Cisalpino. 2008. Cerebral infection

caused by Cryptococcus gattii: a case report and antifungal susceptibility testing.

Rev Iberoam Micol 25:242-5.

63. Sorvillo, F., G. Beall, P. A. Turner, V. L. Beer, A. A. Kovacs, and P. R. Kerndt.

1997. Incidence and factors associated with extrapulmonary cryptococcosis among

persons with HIV infection in Los Angeles County. Aids 11:673-9.

64. Speed, B., and D. Dunt. 1995. Clinical and host differences between infections

with the two varieties of Cryptococcus neoformans. Clin Infect Dis 21:28-34;

discussion 35-6.

65. Springer, D. J., and V. Chaturvedi. 2010. Projecting global occurrence of

Cryptococcus gattii Emerg Infect Dis 16:14-20.

66. Staib, F. 1963. [Membrane filtration and Guizotia abyssinica culture media for the

demonstration of Cryptococcus neoformans (Brown Color Effect).]. Z Hyg

Infektionskr 149:329-36.

67. Swinne, D., J. B. Nkurikiyinfura, and T. L. Muyembe. 1986. Clinical isolates of

Cryptococcus neoformans from Zaire. Eur J Clin Microbiol 5:50-1.

68. Van Dam, M. G., R. A. Seaton, and A. J. Hamilton. 1998. Analysis of HLA

association in susceptibility to infection with Cryptococcus neoformans var. gattii

in a Papua New Guinean population. Med Mycol 36:185-8.

69. Vanbreuseghem, R., and M. Takashio. 1970. An atypical strain of Cryptococcus

neoformans (San Felice) Vuillemin 1894. II. Cryptococcus neoformans var. gattii

var. nov. Ann Soc Belg Med Trop 50:695-702.

70. Varma, A., and K. J. Kwon-Chung. 2010. Heteroresistance of Cryptococcus

gattii to Fluconazole. Antimicrob. Agents Chemother. 54:2303-2311.

3c. Nature, Availability & Source of Reagents/Samples:

Genomic DNAs from reference strains for high quality sequencing.

The Mycology Laboratory at the Wadsworth Center, Albany, NY has extensive experience

with genes and genomics of C. gattii including sequencing of large contigs (Biochemical

and Biophysical Research Communications. 2004: 326, 233–241). Many of the reference

strains to be sequenced are already in this laboratory and stored under liquid nitrogen.

Remaining strains are being procured from collaborating institutions. High quality

genomic DNA will be made in Albany and quality checked before sequencing at the J.

Craig Venter Institute.

C. gattii nucleic acids and other reagents source

The interactive group of principal investigators represents US, Europe, Australia, Africa

and South America. The interests and strengths of these laboratories are in clinical

mycology, ecology, molecular pathogenesis, drug resistance and genomics. The

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investigators are:

Vishnu Chaturvedi, New York State Department of Health, Albany, NY

K.J. Kwon-Chung, National Institute of Allergy and Infectious Diseases, Bethesda, MD

Brian Wong, Oregon Health Sciences Center, Portland, OR

John Perfect, Duke University Medical Center, Durham, NC

Joe Heitman, Duke University Medical Center, Durham, NC

Teun Boekhout, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

Thomas Harrison, St. George’s University of London, UK

Wieland Meyer, University of Sydney, Sydney Australia

Tania Sorell, University of Sydney, Australia

M.H. Veinstein, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

H.F. Vismer, PROMEC Unit, Medical Research Council, Tygerberg, South Africa

C. gattii RNAs will be provided by the following collaborators on a schedule to be

developed.

Vishnu Chaturvedi, New York State Department of Health, Albany, NY

K.J. Kwon-Chung, National Institute of Allergy and Infectious Diseases, Bethesda, MD

Brian Wong, Oregon Health Sciences Center, Portland, OR

John Perfect, Duke University Medical Center, Durham, NC

Joe Heitman, Duke University Medical Center, Durham, NC

Wieland Meyer, University of Sydney, Sydney Australia

M.H. Veinstein, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

3d. Proposed Methods and Protocols to Prepare Reagents:

Genomic DNAs from reference strains.

Standard methods for high quality genomic DNA preparations of C. gattii are available

and used routinely in the laboratories of the participating PIs.

C. gattii RNAs

Total RNA preparations and enrichment for mRNA will be carried out with Qiagen RNA

extraction kits or similar commercial products. All samples will be quality checked for

Illumina RNA-seq application. The source of RNA samples will be different in vitro, in

vivo, ex vivo and ex planta conditions used in participating laboratories to study various

facets of C. gattii in its natural habitat and in human and animal infections.

4. Approach to Data Production:

Reference strain genome sequencing. The proposed sequencing and annotation of 12 C. gattii strains would fill-in gaps in

knowledge from the ongoing efforts on genome sequencing of two strains. The end

product would ensure coverage of all C. gattii serotypes, genotypes, mating types, and

clinical and environmental samples. High resolution genome coverage will be provided by

using multiple runs of short-read next generation sequencing technology. The availability

of reference C. gattii genomes would facilitate fast and accurate assembly and annotation

of the new genomes.

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RNA-seq

Seven collaborating laboratories will provide total RNA from 8 C. gattii strains selected

for functions studies. The conditions of fungal growth will mimic natural habitat, growth

under stress and during various stages of infection in the animal models. The respective

laboratories will use whole genome sequences to analyze expression profiles generated

under various conditions. Eventually,

5. Community Support and Collaborator Roles:

Cryptococcus gattii community. The C. gattii community is currently small and also

includes researchers who study cryptococcosis caused by C. neoformans. However,

clinical and research interest in C. gattii is growing rapidly as exemplified by the coverage

of relevant topics in peer-reviewed and popular press. A specialized international meeting

(Cryptococcus and cryptococcosis) is organized every four years to bring together

interested parties. This meeting attracts from 200 -600 participants hailing from all parts

of the world including researchers who work exclusively with C. gattii. Nevertheless, it is

accepted that much more needs to be done to expand the resources and the researchers

currently working on C. gattii cryptococcosis vis-à-vis C. neoformans cryptococcosis. The

White Paper draft process has allowed a number of investigators to work together to

develop consensus on the existing gaps and future needs in this area.

6. Compliance Requirements:

6a. Review NIAID’s Reagent, Data & Software Release Policy:

http://www3.niaid.nih.gov/research/resources/mscs/data.htm

http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-013.html

Accept X Decline

6b. Public Access to Data and Materials:

Strains.

Most of the C. gattii strains used in this project are available from a number of recognized

culture collections. Similarly, the remaining few strains will be deposited at the NIAID

sponsored BEI at ATCC.

Annotated reference genomes.

C. gattii genomes sequenced in this project, their annotations including the RNA-seq and

sRNA-seq data will be deposited to GenBank at NCBI.

Expression profiling data.

All information pertaining to mRNA and small RNA expression profiling including

descriptions of the biological samples, processed data files, and transcription levels for

each gene will be uploaded to the NCBI’s GEO database.

Sequence reads.

All original short read sequence reads will be deposited to the NCBI's Short Read Archive

sequence database.

http://www3.niaid.nih.gov/research/resources/mscs/data.htm

http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-013.html

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17

6c. Internal Review Board (IRB) / IACUE Yes No X

NIAID approval of IRB documentation is required prior to commencement of work.

No human subjects review is required as all C. gattii strains were obtained from strain

collections and are devoid of any patient identifiers.

Investigator Signature: [electronically signed] Vishnu Chaturvedi

Investigator Name: Vishnu Chaturvedi Date: August 7, 2010

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Genomic Sequencing Centers for Infectious Diseases: Management Plan Form 18

Step II: Development of the Management Plan (This section is to be completed upon approval of the white paper and should be done with the assigned

GSC project leader in conjunction with the participating/collaborating scientists.

Rules of Engagement on Project Approval

1) All sample acquisition will require a MTA and IBC approval.

2) Samples derived from human subjects and / or animals require IRB or IACUC approvals

respectively prior to initiating work.

3) Samples acquired from outside the United States will have to adhere to import/export

regulations.

4) Participants are required to comply with the NIAID data & reagent sharing and dissemination

policy. <provide url>

5) Abstracts, Posters and Manuscripts in preparation should be submitted to NIAID for review

prior to submission. Publications should acknowledge and cite the NIAID contract: “This

project has been funded in whole or part with federal funds from the National Institute of Allergy

and Infectious Diseases, National Institutes of Health, Department of Health and Human

Services under contract number HHSN272200900007C.”

Please Note: This is a draft template. Users may have to adapt contents to a final template NIAID develops in the coming weeks and adhere to a process that will be established for submission and approval of projects; it shall be communicated once finalized.

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Management Plan

Project Title:

Date (MM/DD/YY):

GSC Project Leader

Name

Position

Institution

Address

State

ZIP Code

Telephone

Fax

E-Mail

Project Approach:

Outline the experimental design / strategy. This needs to be within the scope as well as service

offerings / capabilities available at the GSC.

Deliverables:

Describe the deliverables to be generated from the project, including reagents, data and / or

software. Researchers are required to be aware of the Data release/sharing and IP policy of the

Center.

Project Timeline:

Estimate the duration of the project and list the milestones and deliverables with due dates.

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Research Compliance Documentation Requirements:

(If required please attach completed/ approval documentation)

1. Material Transfer Agreement Yes No

Please attach the completed UBMTA or the participating institutions MTA.

http://www.autm.net/Content/NavigationMenu/TechTransfer/TechnologyTransferResources/UBMTA

.htm

2. Internal Review Board (IRB) / IACUE Yes No

Please attach the IRB/IACUE application submitted at the institution where the samples were

collected and prepared, the protocol description, consent form along with the approval letter from

the participating institution(s).

3. Institutional Biosafety Committee Yes No

IBC approval is a requirement to commence the project. <Provide contact information for

Institutional IBC/ Environmental health and Safety officer’s contact information >

4. Other permit requirements based on the project / samples under study

To be procured as relevant by the GSC. <Provide contact person>

a. CDC permit Yes No

b. USDA permit Yes No

c. Import/Export Authorization Yes No

http://www.autm.net/Content/NavigationMenu/TechTransfer/TechnologyTransferResources/UBMTA.htm

http://www.autm.net/Content/NavigationMenu/TechTransfer/TechnologyTransferResources/UBMTA.htm

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Detailed Budget for Proposed Project in US Dollars

(Include all costs to be incurred by the Center including sample preparation and shipping as well as

acquisition of permits, if applicable. Note: The project may incur additional expenses at other

participating institutions which are not included here.)

Budget Item Cost

Total Estimated Costs US $

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NIAID Project Officer Approval:

Maria Y. Giovanni, Ph.D.

Assistant Director for Microbial Genomics & Advanced Technology

Division of Microbiology and Infectious Diseases

NIAID/NIH/DHHS

6610 Rockledge Dr. MSC 6603; Room 6007

Bethesda, MD 20892-6603

301-496-1884 Phone

301-480-4528 FAX

[email protected]

NIAID Contract Officer Approval:

Robert Singman

MID Research Contracts Branch A

Office of Acquisitions, DEA, NIAID, NIH, DHHS

6700B Rockledge Drive

Room 3153, MSC 7612

Bethesda, MD 20892-7612 (FedEx use 20817)

301-451-2607 Phone

301-480-4675 FAX

Participating Institution Business/Administrative Contact:

(Required if there are financial transactions between the participating institutions)

Name

Position

Institution

Address

State

ZIP Code

Telephone

Fax

E-Mail

Participating Institution Business/Administrative Approval:

mailto:[email protected]

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GSC Principal Investigator’s Acceptance and Approval:

Name

Position

Institution

Address

State

ZIP Code

Telephone

Fax

E-Mail

Signature:

GSC Research Administrative Acceptance and Approval

Name

Position

Institution

Address

State

ZIP Code

Telephone

Fax

E-Mail

Signature:

GSC Project Manager’s Contact:

Name

Position

Institution

Address

State

ZIP Code

Telephone

Fax

E-Mail

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